Sperm must have purposeful forward motion following deposition in the female reproductive tract in order to fertilize ovulated eggs. The factors that control the sperm flagellar power stroke and waveform are poorly understood. Spag6, a protein containing eight armadillo repeats, a protein interaction motife, is the murine orthologue of Chlamydomonas PF16, a central apparatus protein required for algae flagellar function. Mice lacking Spag6 are infertile due to a severe sperm motility defect, resulting in part from disruption of the flagellar axoneme architecture. This model will be used to identify proteins that interact with Spag6 and thus expand the catalogue of the mammalian axonemal proteins that are essential for flagellar structure and function. Using two dimensional gel electrophoresis and surface-enhanced laser desorption/ionization mass spectrometry, the proteome of Spag6-deficient sperm will be characterized and compared with wild-type mouse sperm to identify missing proteins. The missing proteins will be characterized and their cDNAs cloned for further documentation of interaction with Spag6 using yeast two-hybrid and pull-down assays and immunocytochemical co-localization. The domains of Spag6 that mediate protein-protein interactions will be defined using yeast two-hybrid and other assays. The proteome of human sperm with motility and ultrastructural defects that mirror those of the Spag6-deficient mouse will be examined to screen for humans with SPAG6 deficiency. The knowledge gained from this research will provide a molecular framework for understanding sperm motility defects that cause male infertility and possibly offer new avenues for contraception through the disruption of purposeful sperm motion.